Cyclic AMP (CAMP) and calcium ion are two key second messengers that transmit numerous extracellular and intracellular signals to control a plethora of physiological responses such as learning and memory, and control of heart rate. There is significant crosstalk between two signals transduced by these two second messengers. One of the intersection point involves calmodulin, a calcium sensor mediator that can activate two classes of adenylyl cyclase, the enzyme that synthesizes cAMP. One class is a toxin secreted from pathogenic bacteria such as edema factor from Bacillus anthracis and the second is adenylyl cyclase from higher eukaryotes such as mammalian type 1 enzyme (AC1). The long-term goal of this project is to elucidate the molecular mechanism that underlies the regulation of bacterial and mammalian adenylyl cyclases by calmodulin. Edema factor consists of two functional domains. The N-terminal portion (28 kDa) mediates association with protective antigen, a transporter produced by B. anthracis so that edema factor can be transported into eukaryotic cell. The C-terminal portion (60 kDa) of edema factor has high adenylyl cyclase activity (the turn over number is around 1,000 per sec) and the activity is highly dependent on calmodulin. We have expressed and purified the C-terminal catalytic domain of edema factor and have obtained diffracting crystals of edema factor alone and in complex with calmodulin. We propose to determine the molecular structures of both forms of the enzyme. We will then use these structures to generate a detailed catalytic model of edema factor activation. We will test this model with biochemical, spectroscopic, and additional crystallographic analyses. We will also use structure-based and genetic- based inhibitor screens to search for the high-affinity small molecules and peptides that block calmodulin activation and catalysis of edema factor. All mammalian membrane-bound adenylyl cyclases share a common structure, including two highly conserved domains (C1a and C2a) connected by the less conserved C1b and transmembrane domains. C1a and C2a form a soluble enzyme that can be activated by the alpha subunit of Gs. C1b region of AC1 consists of an amphipathic, alpha-helical region that is necessary for calmodulin activation. Mutational analysis suggests that activation of AC1 by calmodulin is distinctly different from that of edema factor. We propose to construct a calmodulin-sensitive soluble enzyme using C 1 and C2 domains of AC1 and its homologs. We will analyze calmodulin activates of the soluble AC1 in a manner similar to our analyses of edema factor. Success in this research will not only enhance our knowledge of how adenylyl cyclase is regulated, but also provide important structural insights into how calmodulin modulates the activities of its many other target proteins. In addition, success in finding a lead compound that inhibits edema factor would provide the means to develop better drugs to defend against the infection of B. anthracis.